Overexpression Of Connective Tissue Growth Factor Research Articles

MicroRNA-19 upregulation attenuates cardiac fibrosis via targeting connective tissue growth factor

BackgroundPrevious studies have shown the role of microRNA (miR)-19 in aging-related heart failure. The present study aimed to verify the effects of miR-19 on cardiac fibrosis and its target. MethodsCardiac fibrosis was induced by myocardial infarction (MI)-induced heart failure and angiotensin (Ang) II-treated rats in vivo, and was induced in Ang II-treated cardiac fibroblasts (CFs) in vitro. ResultsThe expression of miR-19 was reduced in the heart tissue of MI and Ang II-treated rats, and Ang II-treated CFs. The impaired cardiac function in rats was repaired after miR-19 administration. The levels of collagen I, collagen III and transforming growth factor-beta (TGF-β) increased in the heart tissue of MI and Ang II-treated rats, and Ang II-treated CFs. These increases were reversed by miR-19 agomiR. Moreover, the bioinformatic analysis and luciferase reporter assays demonstrated that connective tissue growth factor (CTGF) was a direct target of miR-19. MiR-19 treatment inhibited CTGF expression in CFs, while CTGF overexpression inhibited miR-19 agomiR to attenuate the Ang II-induced increases of collagen I and collagen III in CFs. The increases of p-ERK, p-JNK and p-p38 in the CFs induced by Ang II were repressed by miR-19 agomiR. ConclusionsUpregulating miR-19 can improve cardiac function and attenuate cardiac fibrosis by inhibiting the CTGF and MAPK pathways.

Connective tissue growth factor mediates bone morphogenetic protein 2-induced increase in hyaluronan production in luteinized human granulosa cells

BackgroundHyaluronan is the main component of the cumulus-oocyte complex (COC) matrix, and it maintains the basic structure of the COC during ovulation. As a member of the transforming growth factor β (TGF-β) superfamily, bone morphogenetic protein 2 (BMP2) has been identified as a critical regulator of mammalian folliculogenesis and ovulation. However, whether BMP2 can regulate the production of hyaluronan in human granulosa cells has never been elucidated.MethodsIn the present study, we investigated the effect of BMP2 on the production of hyaluronan and the underlying molecular mechanism using both immortalized (SVOG) and primary human granulosa-lutein (hGL) cells. The expression of three hyaluronan synthases (including HAS1, HAS2 and HAS3) were examined following cell incubation with BMP2 at different concentrations. The concentrations of the hyaluronan cell culture medium were determined by enzyme-linked immunosorbent assay (ELISA). The TGF-β type I receptor inhibitors (dorsomorphin and DMH-1) and small interfering RNAs targeting ALK2, ALK3, ALK6 and SMAD4 were used to investigate the involvement of TGF-β type I receptor and SMAD-dependent pathway.ResultsOur results showed that BMP2 treatment significantly increased the production of hyaluronan by upregulating the expression of hyaluronan synthase 2 (HAS2). In addition, BMP2 upregulates the expression of connective tissue growth factor (CTGF), which subsequently mediates the BMP2-induced increases in HAS2 expression and hyaluronan production because overexpression of CTGF enhances, whereas knockdown of CTGF reverses, these effects. Notably, using kinase inhibitor- and siRNA-mediated knockdown approaches, we demonstrated that the inductive effect of BMP2 on the upregulation of CTGF is mediated by the ALK2/ALK3-mediated SMAD-dependent signaling pathway.ConclusionsOur findings provide new insight into the molecular mechanism by which BMP2 promotes the production of hyaluronan in human granulosa cells.

Connective tissue growth factor expression hints at aggressive nature of colorectal cancer

BACKGROUNDConnective tissue growth factor (CTGF) is a mediator of transforming growth factor-beta signaling and plays a key role in connective tissue remodeling, inflammatory processes and fibrosis in various illnesses including cancer. AIMTo investigate the role of CTGF in colorectal cancer (CRC) progression and to compare the CTGF expression with different clinicopathological parameters. METHODSReal-time polymerase chain reaction, immunohistochemistry and Western blotting was performed to evaluate the CTGF expression and the results were statistically analyzed against the clinicopathological variables of patient data using STATA software version 16. RESULTSCTGF expression levels in tumor specimens were significantly higher than their paired normal specimens. The higher protein expression levels showed a significant association with smoking, staging, tumor grade, invasion depth, necrosis of tumor tissue, and both lymphovascular and perineural invasion. As per the cox regression model and classification tree analysis, tumor-node-metastasis stage and perineural invasion were important predictors for CTGF expression and prognosis of CRC patients. Survival analysis indicated that CTGF overexpression was associated with poorer overall and disease-free survival.CONCLUSIONExpression of CTGF was increased in CRC and was linked with poor overall and disease-free survival of CRC patients. These findings support prior observations and thus CTGF may be a possible prognostic marker in CRC.

Molecular laterality encodes stress susceptibility in the medial prefrontal cortex

Functional lateralization of the prefrontal cortex has been implicated in stress and emotional disorders, yet underlying gene expression changes remains unknown. Here, we report molecular signatures lateralized by chronic social defeats between the two medial prefrontal cortices (mPFCs). Stressed mice show 526 asymmetrically expressed genes between the mPFCs. This cortical asymmetry selectively occurs in stressed mice with depressed social activity, but not in resilient mice with normal behavior. We have isolated highly asymmetric genes including connective tissue growth factor (CTGF), a molecule that modulates wound healing at the periphery. Knockdown of CTGF gene in the right mPFC by shRNA led to a stress-resistant behavioral phenotype. Overexpression of CTGF in the right mPFC using viral transduction induces social avoidance while the left mPFC thereof prevent stress-induced social avoidance. Our study provides a molecular window into the mechanism of stress-induced socioemotional disorders, which can pave the way for new interventions by targeting cortical asymmetry.

Regulation of connective tissue growth factor expression by miR-133b for the treatment of renal interstitial fibrosis in aged mice with unilateral ureteral obstruction

IntroductionRenal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.MethodsWe performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.ResultsmiR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70–100 times) in mouse kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.ConclusionmiR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

Prenatal caffeine exposure caused H-type blood vessel-related long bone dysplasia via miR375/CTGF signaling.

Caffeine has developmental toxicity. Prenatal caffeine exposure (PCE) caused intrauterine growth retardation (IUGR) and multiple organ dysplasia. This study intended to explore the effect and mechanism of PCE on long bone development in female fetal rats. In vivo, the PCE group pregnant rats were given different concentrations of caffeine during the gestational Day 9-20. The mRNA expression of osteogenesis-related genes were significantly reduced in PCE group. In the PCE group (120mg/kg·d), the length and primary center of fetal femur were shorter, and accompanied by H-type blood vessel abundance reducing. Meanwhile, connective tissue growth factor (CTGF) expression decreased in the growth plate of the PCE group (120mg/kg·d). In contrast, the miR375 expression increased. In vitro, caffeine decreased CTGF and increased miR375 expression in fetal growth plate chondrocytes. After co-culture with caffeine-treated chondrocytes, the tube formation ability for the H-type endothelial cells was decreased. Furthermore, CTGF overexpression or miR375 inhibitor reversed caffeine-induced reduction of tube formation ability, and miR375 inhibitor reversed caffeine-induced CTGF expression inhibition. In summary, PCE decreased the expression of CTGF by miR375, ultimately resulting in H-type blood vessel-related long bone dysplasia.

MiR-133a-3p inhibits scar formation in scalded mice and suppresses the proliferation and migration of scar derived-fibroblasts by targeting connective tissue growth factor.

Excessive scar formation post burn injury can cause great pain to the patients. MiR-133a-3p has been demonstrated to be anti-fibrotic in some fibrosis-related diseases. However, its possible role in scar formation has not been elucidated yet. In present study, the effect of miR-133a-3p on scar formation was investigated in a scalded model of mice. Moreover, the function of miR-133a-3p on proliferation and migration of scar-derived fibroblasts (SFs) was studied in vitro. It was found that miR-133a-3p was dramatically downregulated in scar tissue of scalded mice. Upregulation of miR-133a-3p by miR-133a-3p agomir obviously inhibited the scar formation in scalded mice. Histological staining showed that upregulation of miR-133a-3p attenuated the excessive deposition of collagen in scar tissue of scalded mice. In vitro study showed that upregulation of miR-133a-3p effectively suppressed the proliferation and migration of SFs. Besides, upregulation of miR-133a-3p attenuated the protein levels of α-smooth muscle actin (α-SMA) and collagen I, indicating that miR-133a-3p could suppress the activation of SFs. The expression of connective tissue growth factor (CTGF), a critical mediator in cell proliferation, migration and extracellular matrix (ECM) synthesis, was also downregulated by the upregulation of miR-133a-3p. Luciferase reporter assay validated that CTGF was directly targeted by miR-133a-3p. In addition, overexpression of CTGF abolished the effect of miR-133a-3p on inhibiting the proliferation, migration and activation of SFs, indicating that miR-133a-3p functioned by targeting CTGF. Therefore, miR-133a-3p might be a promising target for treating pathological scars.

MiR‑26a‑5p alleviates lipopolysaccharide‑induced acute lung injury by targeting the connective tissue growth factor.

The aim of the present study was to investigate the regulatory functions of microRNA (miR)-26a-5p on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and its molecular mechanisms. The role of miR-26a-5p on an ALI mouse model was evaluated by examining the histological changes, wet/dry (W/D) ratio, myeloperoxidase (MPO) activity, malondialdehyde (MDA) expression levels in lung tissues and the survival of ALI mice. Moreover, the protein concentration and the number of neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) was analyzed. To explore the effect of miR-26a-5p on inflammatory responses and apoptosis, the expression levels of tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 and apoptosis were measured by ELISA, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling staining and flow cytometry in BALF, A549 cells and lung tissues. B-cell lymphoma-2 (Bcl-2), Bax and cleaved caspase-3 in lung tissues were measured by western blotting and reverse transcription-quantitative PCR. Connective tissue growth factor (CTGF) was predicted as a direct target of miR-26a-5p using dual luciferase reporter assay. The present study sought to determine whether CTGF overexpression reversed the effect of miR-26a-5p on apoptosis and inflammatory responses in LPS-induced A549 cells. The data revealed that miR-26a-5p overexpression ameliorated LPS-induced ALI, which was implicated by fewer histopathological changes, W/D ratio, apoptosis in lung tissues and the survival of ALI mice. Moreover, miR-26a-5p overexpression alleviated LPS-induced inflammatory responses in ALI mice via the reduction of total protein, neutrophil and lymphocyte counts and the expression levels of TNF-α, IL-1β, IL-6, MDA and MPO activity in BALF. Similarly, miR-26a-5p overexpression decreased apoptosis and the expression of TNF-α, IL-1β and IL-6 in LPS-induced A549 cells. CTGF was a direct target of miR-26a-5p. CTGF overexpression reversed the effect of miR-26a-5p on cell apoptosis and inflammatory responses in LPS-induced A549 cells. The present study demonstrated that miR-26a-5p could attenuate lung inflammation and apoptosis in LPS-induced ALI by targeting CTGF.

Forkhead box K1 facilitates growth of papillary thyroid carcinoma cells by regulating connective tissue growth factor expression.

Forkhead box (FOX) proteins have been identified as key transcription factors in diverse biological processes involved in tumor progression. A large number of FOX proteins are implicated in tumorigenesis of papillary thyroid carcinoma (PTC). Here we investigated the role of Forkhead box K1 (FOXK1) in PTC progression. First, we found that FOXK1 was elevated in both PTC tissues (N = 68) and cell lines. Moreover, up-regulated FOXK1 was associated with shorter overall survival of PTC patients. Second, in vitro functional assays showed that FOXK1 promoted progression of PTC. Mechanistically, FOXK1 could bind to the promoter of cysteine-rich angiogenic inducer 61 (CYR61) and regulate connective tissue growth factor (CTGF) expression through CYR61. Notably, over-expression of CTGF weakened suppression of PTC progression induced by FOXK1 knockdown. Finally, in vivo xenotransplant tumor model indicated that knockdown of FOXK1 suppressed PTC growth. In conclusion, our results indicate that FOXK1 exerts oncogenic roles in PTC via CYR61/CTGF axis, which suggests FOXK1 might act as a potential therapeutic target.

Baicalein alleviated TGF β1-induced type I collagen production in lung fibroblasts via downregulation of connective tissue growth factor

Although we have reported that baicalein ameliorated bleomycin-induced pulmonary fibrosis in rats and inhibited fibroblast-to-myofibroblast differentiation, the mechanisms of the capability of baicalein to suppress the production of type I collagen in fibroblasts remains unclear. Here, we showed that baicalein suppressed transforming growth factor β1 (TGF β1)-stimulated the production of type I collagen in lung fibroblast MRC-5 cells. By applying SILAC-based proteomic technology, 158 proteins were identified as baicalein-modulated proteins in TGF β1-stimulated the accumulation of type I collagen in MRC-5 cells. Our proteomic and biochemical analysis demonstrated that baicalein decreased the expression levels of connective tissue growth factor (CTGF) in TGF β1-stimulated MRC-5 cells. In addition, CTGF overexpression elevated the levels of type I collagen in baicalein-treated fibroblasts. Moreover, our results demonstrated that baicalein-downregulated CTGF expression might be related with the decrease of Smad2 phosphorylation, but not SP1. This work not only linked CTGF to TGF β1-stimulated the production of type I collagen in its attribution to the effects of baicalein, but also might provide valuable information for enhancing the knowledge of the pharmacological inhibition of collagen production, which might represent a promising strategy for the treatment of pulmonary fibrosis.

Long non-coding RNA H19 contributes to wound healing of diabetic foot ulcer.

Diabetic foot ulcer (DFU) is a chronic and non-healing complication of diabetes that leads to high hospital costs and, in extreme cases, to amputation. Recent studies have reported that long non-coding RNAs (lncRNAs) are linked to various diabetes-related symptoms. Thus, we aim to explore the role of lncRNA H19 in the wound healing process following DFU. Fibroblasts were isolated from the ulcer margin tissues of DFU patients, with the expression of lncRNA H19, connective tissue growth factor (CTGF) or serum response factor (SRF) altered by lentivirus infection. Next, rat models of DFU induced by high glucose and lipid diet were established, which was also infected with corresponding lentivirus. The interaction among lncRNA H19, SRF and CTGF was determined. Afterwards, cell proliferation and apoptosis, angiogenesis, ECM remodeling and wound healing in DFU tissues were evaluated to explore the effects of lncRNA H19/SRF/CTGF and MAPK signaling pathway on DFU. CTGF was poorly expressed in ulcer tissues from DFU rats and patients. CTGF overexpression was shown to activate the MAPK signaling pathway to promote cell proliferation, ECM remodeling, angiogenesis and wound healing while inhibiting cell apoptosis. LncRNA H19 was validated to elevate CTGF expression by recruiting SRF to the promoter region of CTGF, thus accelerating cell proliferation, ECM remodeling and wound healing while repressing cell apoptosis. Furthermore, MAPK signaling pathway activation is confirmed to be the underlying mechanism behind lncRNA H19/CTGF/SRF-induced results. Thus, lncRNA H19 accelerated wound healing in DFU through elevation of CTGF and activation of the MAPK signaling pathway.

MiR-26a-5p mediates TLR signaling pathway by targeting CTGF in LPS-induced alveolar macrophage.

To explore the regulation mechanism of miR-26a-5p and connective tissue growth factor (CTGF) in lipopolysaccharide (LPS)-induced alveolar macrophages, which is a severe pneumonia cell model. MH-S cells were grouped into Normal group, Model group, negative control (NC) group, miR-26a-5p mimic group, oe-CTGF group, miR-26a-5p mimic + oe-CTGF group. The expression level of miR-26a-5p, CTGF and Toll-like receptor (TLR) signaling related molecules (TLR2, TLR4 and nuclear factor-κB p65) were detected by qRT-PCR and WB, respectively. The cell viability and apoptosis rate were detected by methyl thiazolyl tetrazolium (MTT) and flow cytometry, respectively. Compared with the Normal group, the expression level of miR-26a-5p was significantly decreased, while CTGF protein level was significantly increased in the Model group. Compared with the Model group, MH-S cells with miR-26a-5p overexpression showed enhanced cell viability, decreased apoptosis rate, declined expression level of TLR signaling related molecules and reduced level of tumor necrosis factor-α (TNF-α), interleukin (IL) 6 (IL-6) and IL-1β, while those with CTGF overexpression had an opposite phenotype. In conclusion, miR-26a-5p can inhibit the expression of CTGF and mediate TLR signaling pathway to inhibit the cell apoptosis and reduce the expression of proinflammatory cytokines in alveolar macrophages which is a cell model of severe pneumonia.

MicroRNA-338-5p alleviates cerebral ischemia/reperfusion injury by targeting connective tissue growth factor through the adenosine 5'-monophosphate-activated protein kinase/mammalian target of rapamycin signaling pathway.

Cerebral ischemia/reperfusion (CIR) injury could lead to the function of brain cell disorder and cerebral infarction. MicroRNAs (miRNAs) have been reported to participate in the progression and protection of CIR injury. Thus, our study aimed to investigate the functional effects of microRNA-338-5p (miR-338-5p) on proliferation, apoptosis, and inflammatory response of CIR injury. According to the results, miR-338-5p was downregulated in the brain of the mice caused by CIR injury, and overexpression of miR-338-5p reduced the neurological deficit and infarct volume of the brain in the mice caused by CIR injury. Meanwhile, miR-338-5p overexpression promoted the proliferation, while suppressed the apoptosis and the inflammatory response of Neuro-2a cells exposed to hypoxia/reoxygenation (H/R). Interestingly, miR-338-5p directly targeted connective tissue growth factor (CTGF) and overexpression of CTGF reversed the functional effects of miR-338-5p on proliferation, apoptosis, and inflammatory response in Neuro-2a cells caused by H/R. More importantly, miR-338-5p affected the adenosine 5¢-monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway by regulating CTGF expression in Neuro-2a cells exposed to H/R. Taken together, we concluded that MiR-338-5p promoted the proliferation, while suppressed the apoptosis and the inflammatory response of cells exposed to H/R by targeting CTGF through the AMPK/mTOR signaling pathway.

Gemcitabine/Nab-Paclitaxel and Pamrevlumab in Locally Advanced Pancreatic Cancer (LAPC): A Phase 1/2 Trial

Background: Pancreatic ductal adenocarcinomas (PDAC) exhibit a high degree of desmoplasia with extensive connective tissue growth factor (CTGF) expression and extracellular matrix production. Tumor response and potential for resection in locally advanced unresectable pancreatic cancer (LAPC) may be affected by pamrevlumab via improved antitumor effect and/or by affecting tumor margins through inhibition of CTGF overexpression. Methods: This is Phase I/II safety/efficacy study in locally advanced pancreatic patients. 37 patients were randomized 2:1 to gemcitabine/nab-paclitaxel plus pamrevlumab (Arm A, n=24) or gemcitabine/nab-paclitaxel alone (Arm B, n=13). Those who completed 6 cycles of treatment were assessed for surgical eligibility, and if eligible, underwent surgical exploration for resection. Patients who did not finish initial chemotherapy received second-line treatment as per standard of care. The study is registered at ClinicalTrials.gov, with a registry number of NCT02210559. Findings: 75% of patients in Arm A and 54% in Arm B completed 6 cycles of therapy. In Arms A and B, CA 19-9 response, as defined by ≥50% decline from baseline occurred in 65% and 42%, respectively; median decline was 93% versus 78%, respectively. Best objective RECIST response was ~30% in each treatment arm. Sixteen percent of patients were radiographically down-staged by NCCN criteria (5 in Arm A and 1 in Arm B). PET SUVmax declined by ≥30% at end of treatment in 60% of patients overall. PET normalized in 38% vs 23% of patients in Arm A vs Arm B, respectively. PET SUVmax normalization at EOT strongly correlated with eligibility for surgical exploration and resection in Arm A and B. Eligibility for surgical exploration was 71% vs 15%, (p=.0019) and resection was achieved in 33% vs 8% of patients in Arm A vs Arm B (p=0.1193), respectively. Interpretation: Pamrevlumab may be a valuable addition to gemcitabine/nab-paclitaxel neoadjuvant therapy in LAPC without added toxicity. This combination merits evaluation in a larger patient population. Trial Registration Number: The study is registered at ClinicalTrials.gov, with a registry number of NCT02210559. Funding: The study was funded by FibroGen, Inc. Declaration of Interest: VP reports other from Ipsen, other from Novocure, other from OncoMed, other from Tyme, other from Celgene, other from Lilly, other from Aduro, FR I/We declare no competing interests, JW I/We declare no competing interests, MP reports other from Genentech, other from Pfizer, other from Merck, other from Astra Zeneca, other from Medimmune, other from Celldex, personal fees and other from ARMO, personal fees and other from Celgene, other from Boston Biomedical, other from Tesaro, other from Bavarian Nordic, other from Novartis, other from BMS, other from Abbvie, other from Halozyme, other from Karyopharm, other from Bayer, other from Regeneron, personal fees from Sirtex, personal fees from Caris, personal fees from Rafael, personal fees from Eisai, personal fees from Ignyta, personal fees from Ipsen, personal fees from Merrimack, outside the submitted work, KM reports other from Agios, other from Senwah Biosciences, other from Taiho, other from Arqule, other from Astra Zeneca, other from Genentech, other from Incyte, other from Tracon Pharmaceuticals, other from Medimmune, other from Puma Biotechnology, personal fees from Astra Zeneca, personal fees from Bayer, personal fees from Celgene, personal fees from Eisai, personal fees from Exelixis, personal fees from Merrimack, personal fees from Vicus, personal feels from Ipsen, outside the submitted work, JG reports other from Genentech, other from Generon, other from GlaxoSmithKline, other from Hana Biosciences, other from Kyowa, other from Novartis, other from Odonate, other from Pfizer, other from Polynoma, other from Terapure, TL I/We declare no competing interests, MM I/We declare no competing interests, MC is employed by FibroGen, MZ is employed by FibroGen, SP is employed by FibroGen, EK is employed by FibroGen, TN is employed by FibroGen, EC is employed by Fibrogen Ethical Approval: The study was approved by individual institutional review boards at 9 US institutions and conducted according to the Declaration of Helsinki.

Local Silencing of Connective Tissue Growth Factor by siRNA/Peptide Improves Dermal Collagen Arrangements

Collagen organization within tissues has a critical role in wound regeneration. Collagen fibril diameter, arrangements and maturity between connective tissue growth factor (CTGF) small interfering RNA (siRNA) and mismatch scrambled siRNA-treated wound were compared to evaluate the efficacy of CTGF siRNA as a future implement for scar preventive medicine. Nanocomplexes of CTGF small interfering RNA (CTGF siRNA) with cell penetrating peptides (KALA and MPG∆NLS) were formulated and their effects on CTGF downregulation, collagen fibril diameter and arrangement were investigated. Various ratios of CTGF siRNA and peptide complexes were prepared and down-regulation were evaluated by immunoblot analysis. Control and CTGF siRNA modified cells-populated collagen lattices were prepared and rates of contraction measured. Collagen organization in rabbit ear 8mm biopsy punch wound at 1day to 8wks post injury time were investigated by transmission electron microscopy and histology was investigated with Olympus System and TS-Auto software. CTGF expression was down-regulated to 40% of control by CTGF siRNA/KALA (1:24) complexes (p < 0.01) and collagen lattice contraction was inhibited. However, down-regulated of CTGF by CTGF siRNA/MPG∆NLS complexes was not statistically significant. CTGF KALA-treated wound appeared with well formed-basket weave pattern of collagen fibrils with mean diameter of 128 ± 22nm (n = 821). Mismatch siRNA/KALA-treated wound showed a high frequency of parallel small diameter fibrils (mean 90 ± 20nm, n = 563). Controlling over-expression of CTGF by peptide-mediated siRNA delivery could improve the collagen orientation and tissue remodeling in full thickness rabbit ear wound.

Effect of anti-CTGF human recombinant monoclonal antibody pamrevlumab on resectability and resection rate when combined with gemcitabine/nab-paclitaxel in phase 1/2 clinical study for the treatment of locally advanced pancreatic cancer patients.

4016Background: Pancreatic ductal adenocarcinomas exhibit high degree of desmoplasia with extensive connective tissue growth factor (CTGF) expression and extracellular matrix production. CTGF overexpression is associated with aberrant fibrous tissue in mouse model, in which progression of tissue adhesion was inhibited by pamrevlumab. We hypothesize that pamrevlumab, an anti-CTGF antibody, may influence resectability of locally advanced pancreatic cancer (LAPC) by inhibiting effects of CTGF. Methods: Pamrevlumab + gemcitabine/Nab-paclitaxel (G/N) (Arm A) vs G/N (Arm B) was given to treatment-naive LAPC patients to improve resection rate and overall survival (OS). Patients (N = 37) were randomized 2:1 in Arm A vs Arm B. Patients who completed 6 cycles of treatment underwent resectability assessment per protocol criteria (NCCN, CA 19-9, PET, RECIST) and, if found eligible, underwent resection. No adjuvant therapy was given; second line therapy was administered per investigator discretion. Results: In the ITT...

Exosomal miR-214 from endometrial stromal cells inhibits endometriosis fibrosis

Is it possible to improve fibrosis in endometriosis by microRNA-214 delivery in exosomes? Upregulation of miR-214 may inhibit fibrogenesis and its delivery by exosomes derived from ectopic endometrial stromal cells (ESCs), offers an alternative therapeutic approach for endometriosis fibrosis. Fibrosis is the primary pathological feature of endometriosis. MiR-214 plays an important role in fibrotic disease. Connective tissue growth factor (CTGF) is a critical fibrogenic mediator of miR-214. The expression of miR-214 is decreased in ectopic ESCs compared with normal ESCs. miRNAs are a natural cargo of exosomes and these could be exploited as carriers of miRNA in replacement therapy. Paired eutopic and ectopic endometrial tissue samples were obtained from 10 women with ovarian endometrioma. ESCs and epithelial cells from both were cultured in vitro. RT-PCR, western blot and immunohistochemistry were used to study the effect of transfection with miR-214 mimics on CTGF expression and fibrogenesis respectively, with and without TGFβ stimulation. Exosomes were isolated from ectopic ESCs and Endometrioma tissue was isolated from four patients, dispersed an injected (ip) into nude mice and allowed to implant. The mice were treated with miR-214-enriched exosomes or controls to confirm the effect of inhibiting CTGF overexpression on endometriosis fibrosis. The primary ectopic ESCs were transfected with miR-214 mimics. The levels of miR-214, CTGF and fibrotic markers were measured by RT-PCR and Immunohistochemistry. A mouse model of endometriosis was established by ip injection of human ectopic endometrial tissues into nude mice. MiR-214-enriched exosomes were injected into the mice and endometriotic lesions were measured on Day 28. Changes in fibrosis of the endometriotic implants were studied by histopathological staining. CTGF and fibrotic markers upregulation in endometriosis is associated with a reciprocal down-regulation of miR-214. By using miR-214 mimics and antagomirs to investigate expression of fibrotic markers, we found that increased production of miR-214 reduced Collagen αI and CTGF expression in endometriosis stromal and endometrial epithelial cells in response to fibrosis-inducing stimuli (P < 0.001 versus non-treatment). Ectopic ESCs yielded nano-sized exosomes which expressed miR-214. Loading exosomes with miR-214 mimics and injecting them into an experimental endometriosis mouse model resulted in a decrease in the expression of fibrosis-associated proteins (P < 0.001 versus PBS control group). N/A. We only isolated exosomes from ectopic ESCs, whether this is the optimum source requires further study. Upregulation of miRNA-214 potentially offers an alternative therapeutic approach for endometriosis fibrosis. This work was supported by grants from the National Natural Science Foundation of China (Grant no. 81771549 Jinwei Miao). The authors declare that there is no conflict of interest.

MiR-133b regulates the expression of CTGF in epithelial-mesenchymal transition of ovarian cancer.

To explore the role of miR-133b in ovarian cancer and to preliminarily elucidate the mechanism of miR-133b in epithelial-mesenchymal transition (EMT) of ovarian cancer. MiR-133b was detected in ovarian cancer specimens, and the relationship of miR-133b with each pathological index and clinical index of ovarian cancer was analyzed. The action targets of miR-133b in ovarian cancer were analyzed systematically and studied deeply via the target validation and cell function validation. Finally, the possible reasons of ovarian cancer metastasis were analyzed through the molecular regulation mechanism in EMT of ovarian cancer. The miR-133b level in ovarian cancer was significantly lower than in normal ovarian tissues and benign ovarian tumors (p<0.05). The level of miR-133b in ovarian cancer was related to differentiated degree and lymphatic metastasis. Dual-luciferase assay indicated that connective tissue growth factor (CTGF) was the target gene regulated by miR-133b. Reverse transcriptase-polymerase chain reaction (RT-PCR) as well as Western blot results proved that the expression level of E-cadherin representing the epithelial cell phenotype was increased, while the expression level of vimentin representing the mesenchymal cell phenotype was decreased. Transwell assay confirmed that the migration and invasion abilities of ovarian cancer cells declined after transfection with miR-133b plasmid. After co-transfection with miR-133b and CTGF overexpression plasmids, RT-PCR and Western blotting proved that the expression level of E-cadherin representing the epithelial cell phenotype was decreased, while the expression level of vimentin representing the mesenchymal cell phenotype was increased; transwell assay confirmed that the cell migration and invasion abilities were increased after co-transfection. The results of this study showed that miR-133b may serve as a new molecular marker of EMT of ovarian cancer, and act as a molecular marker of differentiated degree and lymphatic metastasis of ovarian cancer.

Synergistic promoting effects of bone morphogenetic protein 12/connective tissue growth factor on functional differentiation of tendon derived stem cells and patellar tendon window defect regeneration

Current study investigated bone morphogenetic protein 12 (BMP12) and connective tissue growth factor (CTGF) activate tendon derived stem cells (TDSCs) tenogenic differentiation, and promotion of injured tendon regeneration. TDSCs were transfected with BMP12 and CTGF via recombinant adenovirus (Ad) infection. Gene transfection efficiency, cell viability and cytotoxicity, tenogenic gene expression, collagen I/III synthesis were evaluated in vitro. For the in vivo study, the transfected cells were transplanted into the rat patellar tendon window defect. At weeks 2 and 8 of post-surgery, the repaired tendon tissues were harvested for histological and biomechanical examinations. The transfected TDSCs revealed relatively stable transfection efficiency (80–90%) with active cell viability means while rare cytotoxicity in each group. During days 1 and 5, BMP12 and CTGF transfection caused tenogenic differentiation genes activation in TDSCs: type I/III collagen, tenascin-C, and scleraxis were all up-regulated, whereas osteogenic, adipogenic, and chondrogenic markers were all down-regulated respectively. In addition, BMP12 and CTGF overexpression significantly promote type I/III collagen synthesis. After in vivo transplantation, at 2 and 8 weeks post-surgery, BMP12, CTGF and co-transfection groups showed more integrated tendon tissue structure versus control, meanwhile, the ultimate failure loads and Young’s were all higher than control. Remarkably, at 8 weeks post-surgery, the biomechanical properties of co-transfection group was approaching to normal rat patellar tendon, moreover, the ratio of type III/I collagen maintained about 20% in each transfection group, meanwhile, the type I collagen were significantly increased with co-transfection treatment. In conclusion, BMP12 and CTGF transfection stimulate tenogenic differentiation of TDSCs. The synergistic effects of simultaneous transfection of both may significantly promoted rat patellar tendon window defect regeneration.

Angiotensin II induces connective tissue growth factor expression in human hepatic stellate cells by a transforming growth factor \u03b2-independent mechanism

Angiotensin II (Ang II) promotes hepatic fibrosis by increasing extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF) plays a crucial role in the pathogenesis of hepatic fibrosis and emerges as downstream of the profibrogenic cytokine transforming growth factor-β (TGF-β). We aimed to investigate the molecular events that lead from the Ang II receptor to CTGF upregulation in human hepatic stellate cells, a principal fibrogenic cell type. Ang II produced an early, AT1 receptor-dependent stimulation of CTGF expression and induced a rapid activation of PKC and its downstream p38 MAPK, thereby activating a nuclear factor-κB (NF-κB) and Smad2/3 cross-talk pathway. Chemical blockade of NF-κB and Smad2/3 signaling synergistically diminished Ang II-mediated CTGF induction and exhibited an additive effect in abrogating the ECM accumulation caused by Ang II. Furthermore, we demonstrated that CTGF expression was essential for Ang II-mediated ECM synthesis. Interestingly, the ability of dephosphorylated, but not phosphorylated JNK to activate Smad2/3 signaling revealed a novel role of JNK in Ang II-mediated CTGF overexpression. These results suggest that Ang II induces CTGF expression and ECM accumulation through a special TGF-β-independent interaction between the NF-κB and Smad2/3 signals elicited by the AT1/PKCα/p38 MAPK pathway.